Publications

2024

1.
Zaferani M, Song R, Petry S, Stone H. Building on-chip cytoskeletal circuits via branched microtubule networks. Proceedings of the National Academy of Sciences of the United States of America. 2024;121(4):e2315992121. PMCID: PMC10823238

Controllable platforms to engineer robust cytoskeletal scaffolds have the potential to create novel on-chip nanotechnologies. Inspired by axons, we combined the branching microtubule (MT) nucleation pathway with microfabrication to develop "cytoskeletal circuits." This active matter platform allows control over the adaptive self-organization of uniformly polarized MT arrays via geometric features of microstructures designed within a microfluidic confinement. We build and characterize basic elements, including turns and divisions, as well as complex regulatory elements, such as biased division and MT diodes, to construct various MT architectures on a chip. Our platform could be used in diverse applications, ranging from efficient on-chip molecular transport to mechanical nano-actuators. Further, cytoskeletal circuits can serve as a tool to study how the physical environment contributes to MT architecture in living cells.

2023

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Gouveia B, Petry S. +TIPs condense on microtubule plus-ends. Nature cell biology. 2023;25(1):12–14. PMID: 36650376
1.
Gouveia B, Setru S, King M, Hamlin A, Stone H, Shaevitz J, Petry S. Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes in Xenopus laevis egg extract. Nature communications. 2023;14(1):3696. PMCID: PMC10284841

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area.

1.
Guo C, Alfaro-Aco R, Zhang C, Russell R, Petry S, Polenova T. Structural basis of protein condensation on microtubules underlying branching microtubule nucleation. Nature communications. 2023;14(1):3682. PMCID: PMC10284871

Targeting protein for Xklp2 (TPX2) is a key factor that stimulates branching microtubule nucleation during cell division. Upon binding to microtubules (MTs), TPX2 forms condensates via liquid-liquid phase separation, which facilitates recruitment of microtubule nucleation factors and tubulin. We report the structure of the TPX2 C-terminal minimal active domain (TPX2) on the microtubule lattice determined by magic-angle-spinning NMR. We demonstrate that TPX2 forms a co-condensate with soluble tubulin on microtubules and binds to MTs between two adjacent protofilaments and at the intersection of four tubulin heterodimers. These interactions stabilize the microtubules and promote the recruitment of tubulin. Our results reveal that TPX2 is disordered in solution and adopts a folded structure on MTs, indicating that TPX2 undergoes structural changes from unfolded to folded states upon binding to microtubules. The aromatic residues form dense interactions in the core, which stabilize folding of TPX2 on microtubules. This work informs on how the phase-separated TPX2 behaves on microtubules and represents an atomic-level structural characterization of a protein that is involved in a condensate on cytoskeletal filaments.

1.
Kalra A, Benny A, Travis S, Zizzi E, Morales-Sanchez A, Oblinsky D, Craddock T, Hameroff S, MacIver B, Tuszyński J, Petry S, Penrose R, Scholes G. Electronic Energy Migration in Microtubules. ACS central science. 2023;9(3):352–361. PMCID: PMC10037452

The repeating arrangement of tubulin dimers confers great mechanical strength to microtubules, which are used as scaffolds for intracellular macromolecular transport in cells and exploited in biohybrid devices. The crystalline order in a microtubule, with lattice constants short enough to allow energy transfer between amino acid chromophores, is similar to synthetic structures designed for light harvesting. After photoexcitation, can these amino acid chromophores transfer excitation energy along the microtubule like a natural or artificial light-harvesting system? Here, we use tryptophan autofluorescence lifetimes to probe energy hopping between aromatic residues in tubulin and microtubules. By studying how the quencher concentration alters tryptophan autofluorescence lifetimes, we demonstrate that electronic energy can diffuse over 6.6 nm in microtubules. We discover that while diffusion lengths are influenced by tubulin polymerization state (free tubulin versus tubulin in the microtubule lattice), they are not significantly altered by the average number of protofilaments (13 versus 14). We also demonstrate that the presence of the anesthetics etomidate and isoflurane reduce exciton diffusion. Energy transport as explained by conventional Förster theory (accommodating for interactions between tryptophan and tyrosine residues) does not sufficiently explain our observations. Our studies indicate that microtubules are, unexpectedly, effective light harvesters.

1.
Kraus J, Travis S, King M, Petry S. Augmin is a Ran-regulated spindle assembly factor. The Journal of biological chemistry. 2023;:104736. PMCID: PMC10318467

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αβ and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. InXenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is non-essential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and TIRF microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-β through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT binding site. Moreover, we show Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.

1.
Kraus J, Alfaro-Aco R, Gouveia B, Petry S. Microtubule nucleation for spindle assembly: one molecule at a time. Trends in biochemical sciences. 2023;48(9):761–775. PMCID: PMC10769297

The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.

1.
Travis S, Mahon B, Huang W, Ma M, Rale M, Kraus J, Taylor D, Zhang R, Petry S. Integrated model of the vertebrate augmin complex. Nature communications. 2023;14(1):2072. PMCID: PMC10102177

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin's structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation.

1.
Valdez V, Neahring L, Petry S, Dumont S. Mechanisms underlying spindle assembly and robustness. Nature reviews. Molecular cell biology. 2023;24(8):523–542. PMCID: PMC10642710

The microtubule-based spindle orchestrates chromosome segregation during cell division. Following more than a century of study, many components and pathways contributing to spindle assembly have been described, but how the spindle robustly assembles remains incompletely understood. This process involves the self-organization of a large number of molecular parts - up to hundreds of thousands in vertebrate cells - whose local interactions give rise to a cellular-scale structure with emergent architecture, mechanics and function. In this Review, we discuss key concepts in our understanding of spindle assembly, focusing on recent advances and the new approaches that enabled them. We describe the pathways that generate the microtubule framework of the spindle by driving microtubule nucleation in a spatially controlled fashion and present recent insights regarding the organization of individual microtubules into structural modules. Finally, we discuss the emergent properties of the spindle that enable robust chromosome segregation.

2022

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Gouveia B, Kim Y, Shaevitz J, Petry S, Stone H, Brangwynne C. Capillary forces generated by biomolecular condensates. Nature. 2022;609(7926):255–264. PMID: 36071192

Liquid-liquid phase separation and related phase transitions have emerged as generic mechanisms in living cells for the formation of membraneless compartments or biomolecular condensates. The surface between two immiscible phases has an interfacial tension, generating capillary forces that can perform work on the surrounding environment. Here we present the physical principles of capillarity, including examples of how capillary forces structure multiphase condensates and remodel biological substrates. As with other mechanisms of intracellular force generation, for example, molecular motors, capillary forces can influence biological processes. Identifying the biomolecular determinants of condensate capillarity represents an exciting frontier, bridging soft matter physics and cell biology.

To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified γTuRCs in vitro (Thawani et al., , 2020; Liu et al., 2020). Here, we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a γTuRC activator. We further validate this effect in egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA's L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA's activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated.

1.
Travis S, Mahon B, Petry S. How Microtubules Build the Spindle Branch by Branch. Annual review of cell and developmental biology. 2022;38:1–23. PMCID: PMC9619725

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

2021

1.
Gai Y, Cook B, Setru S, Stone H, Petry S. Confinement size determines the architecture of Ran-induced microtubule networks. Soft Matter. 2021;17(24):5921–5931. PMCID: PMC8958645

The organization of microtubules (MTs) is critical for cells during interphase and mitosis. During mitotic spindle assembly, MTs are made and organized around chromosomes in a process regulated by RanGTP. The role of RanGTP has been explored in Xenopus egg extracts, which are not limited by a cell membrane. Here, we investigated whether cell-sized confinements affect the assembly of RanGTP-induced MT networks in Xenopus egg extracts. We used microfluidics to encapsulate extracts within monodisperse extract-in-oil droplets. Importantly, we find that the architecture of Ran-induced MT networks depends on the droplet diameter and the Ran concentration, and differs from structures formed in bulk extracts. Our results highlight that both MT nucleation and physical confinement play critical roles in determining the spatial organization of the MT cytoskeleton.

1.
Safari M, King M, Brangwynne C, Petry S. Interaction of spindle assembly factor TPX2 with importins-α/β inhibit protein phase separation. J Biol Chem. 2021;:100998. PMID: 34302807

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from pre-existing ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 mono-dispersed trimer. We also identify a new nuclear localization sequence (NLS) in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

1.
Setru S, Gouveia B, Alfaro-Aco R, Shaevitz J, Stone H, Petry S. A hydrodynamic instability drives protein droplet formation on microtubules to nucleate branches. Nat Phys. 2021;17(4):493–498. PMID: 35211183
1.
Thawani A, Petry S. Molecular insight into how γ-TuRC makes microtubules. Journal of cell science. 2021;134(14). PMCID: PMC8325954

As one of four filament types, microtubules are a core component of the cytoskeleton and are essential for cell function. Yet how microtubules are nucleated from their building blocks, the αβ-tubulin heterodimer, has remained a fundamental open question since the discovery of tubulin 50 years ago. Recent structural studies have shed light on how γ-tubulin and the γ-tubulin complex proteins (GCPs) GCP2 to GCP6 form the γ-tubulin ring complex (γ-TuRC). In parallel, functional and single-molecule studies have informed on how the γ-TuRC nucleates microtubules in real time, how this process is regulated in the cell and how it compares to other modes of nucleation. Another recent surprise has been the identification of a second essential nucleation factor, which turns out to be the well-characterized microtubule polymerase XMAP215 (also known as CKAP5, a homolog of chTOG, Stu2 and Alp14). This discovery helps to explain why the observed nucleation activity of the γ-TuRC in vitro is relatively low. Taken together, research in recent years has afforded important insight into how microtubules are made in the cell and provides a basis for an exciting era in the cytoskeleton field.

2020

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Alfaro-Aco R, Thawani A, Petry S. Biochemical reconstitution of branching microtubule nucleation. Elife. 2020;9. PMID: 31933480

Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways and their mode of action still need to be identified. Using purified proteins we biochemically reconstitute branching microtubule nucleation, which is critical for chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the branching effectors augmin and TPX2 are required to efficiently nucleate microtubules from pre-existing microtubules. TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice. TPX2 and augmin enable γ-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly-spaced patches along microtubules. This work provides a blueprint for other microtubule nucleation pathways and helps explain how microtubules are generated in the spindle.

1.
Amin J, Petry S, Yang H, Shaevitz J. Uniform intensity in multifocal microscopy using a spatial light modulator. PLoS One. 2020;15(3):e0230217. PMID: 32160259
Multifocal microscopy (MFM) offers high-speed three-dimensional imaging through the simultaneous image capture from multiple focal planes. Conventional MFM systems use a fabricated grating in the emission path for a single emission wavelength band and one set of focal plane separations. While a Spatial Light Modulator (SLM) can add more flexibility as a replacement to the fabricated grating, the relatively small number of pixels in the SLM chip, cross-talk between the pixels, and aberrations in the imaging system can produce non-uniform intensity in the different axially separated image planes. We present an in situ iterative SLM calibration algorithm that overcomes these optical- and hardware-related limitations to deliver near-uniform intensity across all focal planes. Using immobilized gold nanoparticles under darkfield illumination, we demonstrate superior intensity evenness compared to current methods. We also demonstrate applicability across emission wavelengths, axial plane separations, imaging modalities, SLM settings, and different SLM manufacturers. Therefore, our microscope design and algorithms provide an alternative to the use of fabricated gratings in MFM, as they are relatively simple and could find broad applications in the wider research community.
1.
King M, Petry S. Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation. Nat Commun. 2020;11(1):270. PMID: 31937751

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/β heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.

1.
Thawani A, Rale M, Coudray N, Bhabha G, Stone H, Shaevitz J, Petry S. The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy. Elife. 2020;9. PMID: 32538784

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

2019

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Petry S. How to run an academic lab based on a basketball strategy. Molecular biology of the cell. 2019;30(23):2859–2861. PMCID: PMC6822588

It is a huge honor to receive the WICB Junior Award for Excellence in Research. In this essay, I first describe what we have discovered in my lab so far. Then, I summarize the strategies that helped transition my lab from its infant shoes to a mature research environment where discoveries are being made.

1.
Thawani A, Stone H, Shaevitz J, Petry S. Spatiotemporal organization of branched microtubule networks. Elife. 2019;8. PMID: 31066674

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.

2018

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Dixit R, Petry S. The life of a microtubule. Mol Biol Cell. 2018;29(6):689. PMID: 29535173
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Rale M, Kadzik R, Petry S. Phase Transitioning the Centrosome into a Microtubule Nucleator. Biochemistry. 2018;57(1):30–37. PMID: 29256606

Centrosomes are self-assembling, micron-scale, nonmembrane bound organelles that nucleate microtubules (MTs) and organize the microtubule cytoskeleton of the cell. They orchestrate critical cellular processes such as ciliary-based motility, vesicle trafficking, and cell division. Much is known about the role of the centrosome in these contexts, but we have a less comprehensive understanding of how the centrosome assembles and generates microtubules. Studies over the past 10 years have fundamentally shifted our view of these processes. Subdiffraction imaging has probed the amorphous haze of material surrounding the core of the centrosome revealing a complex, hierarchically organized structure whose composition and size changes profoundly during the transition from interphase to mitosis. New biophysical insights into protein phase transitions, where a diffuse protein spontaneously separates into a locally concentrated, nonmembrane bounded compartment, have provided a fresh perspective into how the centrosome might rapidly condense from diffuse cytoplasmic components. In this Perspective, we focus on recent findings that identify several centrosomal proteins that undergo phase transitions. We discuss how to reconcile these results with the current model of the underlying organization of proteins in the centrosome. Furthermore, we reflect on how these findings impact our understanding of how the centrosome undergoes self-assembly and promotes MT nucleation.

1.
Song J-G, Petry S. Dissecting Protein Complexes in Branching Microtubule Nucleation Using Meiotic Egg Extracts. Cold Spring Harb Protoc. 2018;2018(9):pdb.prot100958. PMID: 29321281

The mitotic spindle is the microtubule-based apparatus that reliably segregates chromosomes during cell division. Recently, it was discovered that microtubules originate within the mitotic spindle by nucleating off of existing spindle microtubules. This mechanism, termed branching microtubule nucleation, allows the efficient amplification of microtubules while preserving their original polarity as required in the spindle. Three molecular players are known to be involved in this process, namely, the protein TPX2, the protein complex augmin, and the gamma-tubulin ring complex; however, little is known about the assembly of the protein complexes. Here, we use the eight-subunit augmin complex as an example of how to dissect the function and assembly of a protein complex using meiotic egg extracts. Specifically, immunodepletion combined with total internal reflection fluorescence (TIRF) microscopy is used to identify the role of the protein complex. In parallel, immunoprecipitation (IP) and tandem mass spectrometry (MS/MS) are used to infer how it is assembled. This approach can be applied to investigate the assembly of other multisubunit protein complexes that function in branching microtubule nucleation and mitotic spindle assembly.

1.
Song J-G, King M, Zhang R, Kadzik R, Thawani A, Petry S. Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules. J Cell Biol. 2018;217(7):2417–2428. PMID: 29875259

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

1.
Thawani A, Kadzik R, Petry S. XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex. Nat Cell Biol. 2018;20(5):575–585. PMID: 29695792

How microtubules (MTs) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the universal MT nucleator of the cell. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and the γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together, these factors synergistically stimulate MT nucleation in vitro. The amino-terminal TOG domains 1-5 of XMAP215 bind to αβ-tubulin and promote MT polymerization, whereas the conserved carboxy terminus is required for efficient MT nucleation and directly binds to γ-tubulin. In summary, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

2017

1.
Alfaro-Aco R, Thawani A, Petry S. Structural analysis of the role of TPX2 in branching microtubule nucleation. J Cell Biol. 2017;216(4):983–997. PMID: 28264915

The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2's ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.

1.
Alfaro-Aco R, Petry S. How TPX2 helps microtubules branch out. Cell Cycle. 2017;16(17):1560–1561. PMID: 28767311

2016

Mitotic and meiotic spindles consist primarily of microtubules, which originate from centrosomes and within the vicinity of chromatin. Indirect evidence suggested that microtubules also originate throughout the spindle, but the high microtubule density within the spindle precludes the direct observation of this phenomenon. By using meiotic Xenopus laevis egg extract and employing total internal reflection (TIRF) microscopy, microtubule nucleation from preexisting microtubules could be demonstrated and analyzed. Branching microtubule nucleation is an ideal mechanism to assemble and maintain a mitotic spindle, because microtubule numbers are amplified while preserving their polarity. Here, we describe the assays that made these findings possible and the experiments that helped identify the key molecular players involved.
1.
Petry S. Mechanisms of Mitotic Spindle Assembly. Annual review of biochemistry. 2016;85:659–83. PMCID: PMC5016079

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

2015

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Alfaro-Aco R, Petry S. Building the Microtubule Cytoskeleton Piece by Piece. J Biol Chem. 2015;290(28):17154–62. PMID: 25957410

The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of self-organized MT networks in the cell.

1.
Petry S, Vale R. Microtubule nucleation at the centrosome and beyond. Nat Cell Biol. 2015;17(9):1089–93. PMID: 26316453

2013

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Petry S, Groen A, Ishihara K, Mitchison T, Vale R. Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. Cell. 2013;152(4):768–77. PMID: 23415226

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

2011

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Petry S, Vale R. A new cap for kinetochore fibre minus ends. Nat Cell Biol. 2011;13(12):1389–91. PMID: 22081093

In mitotic spindles, each sister chromatid is directly attached to a spindle pole through microtubule bundles known as kinetochore fibres. Microspherule protein 1 (MCRS1) is now shown to support spindle assembly by localizing to the minus ends of kinetochore fibres and protecting them from depolymerization.

1.
Petry S, Pugieux C, Nédélec F, Vale R. Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts. Proc Natl Acad Sci U S A. 2011;108(35):14473–8. PMID: 21844347

Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis. We used an assay system in which hundreds of meiotic spindles can be observed forming around chromatin-coated beads after introduction of Xenopus egg extracts. Spindles forming in augmin-depleted extracts showed reduced rates of MT formation and were predominantly multipolar, revealing a function of augmin in stabilizing the bipolar shape of the acentrosomal meiotic spindle. Our studies also have uncovered an apparent augmin-independent MT nucleation process from acentrosomal poles, which becomes increasingly active over time and appears to partially rescue the spindle defects that arise from augmin depletion. Our studies reveal that spatially and temporally distinct MT generation pathways from chromatin, spindle MTs, and acentrosomal poles all contribute to robust bipolar spindle formation in meiotic extracts.

2009

1.
Uehara R, Nozawa R- suke, Tomioka A, Petry S, Vale R, Obuse C, Goshima G. The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells. Proc Natl Acad Sci U S A. 2009;106(17):6998–7003. PMID: 19369198

The mitotic spindle is constructed from microtubules (MTs) nucleated from centrosomes, chromosome proximal regions, and preexisting spindle MTs. Augmin, a recently identified protein complex, is a critical factor in spindle MT-based MT generation in Drosophila S2 cells. Previously, we identified one subunit of human augmin. Here, by using mass spectrometry, we identified the full human augmin complex of 8 subunits and show that it interacts with the gamma-tubulin ring complex (gamma-TuRC). Unlike augmin-depleted S2 cells, in which the defect in spindle-mediated MT generation is mostly compensated by centrosomal MTs, augmin knockdown alone in HeLa cells triggers the spindle checkpoint, reduces tension on sister kinetochores, and severely impairs metaphase progression. Human augmin knockdown also reduces the number of central spindle MTs during anaphase and causes late-stage cytokinesis failure. A link between augmin and gamma-TuRC is likely critical for these functions, because a gamma-TuRC mutant that attenuates interaction with augmin does not restore function in vivo. These results demonstrate that MT generation mediated by augmin and gamma-TuRC is critical for chromosome segregation and cytokinesis in human cells.

2008

1.
Petry S, Weixlbaumer A, Ramakrishnan. The termination of translation. Curr Opin Struct Biol. 2008;18(1):70–7. PMID: 18206363

Recent results from cryoelectron microscopy, crystallography, and biochemical experiments have shed considerable light on the process by which protein synthesis is terminated when a stop codon is reached. However, a detailed understanding of the underlying mechanisms will require higher-resolution structures of the various states involved.

1.
Weixlbaumer A, Jin H, Neubauer C, Voorhees R, Petry S, Kelley A, Ramakrishnan V. Insights into translational termination from the structure of RF2 bound to the ribosome. Science. 2008;322(5903):953–6. PMID: 18988853

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.

2007

1.
Weixlbaumer A, Petry S, Dunham C, Selmer M, Kelley A, Ramakrishnan. Crystal structure of the ribosome recycling factor bound to the ribosome. Nat Struct Mol Biol. 2007;14(8):733–7. PMID: 17660830

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.

2006

1.
Selmer M, Dunham C, Murphy F, Weixlbaumer A, Petry S, Kelley A, Weir J, Ramakrishnan. Structure of the 70S ribosome complexed with mRNA and tRNA. Science. 2006;313(5795):1935–42. PMID: 16959973

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

2005

1.
Petry S, Brodersen D, Murphy F, Dunham C, Selmer M, Tarry M, Kelley A, Ramakrishnan. Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon. Cell. 2005;123(7):1255–66. PMID: 16377566

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.